Difference between revisions of "Part:BBa K1875005"

Revision as of 05:06, 19 October 2016

A 20 bp target sequence and PAM to make guide operator 1

The [http://2016.igem.org/Team:BostonU BostonU 2016] iGEM team synthesized guide operator sequences composed of a 20 base pair target sequence and a PAM sequence. The target sequence was made using a random DNA generator and entered into the [http://crispr.mit.edu/ CRISPR Design Optimization] tool for the human genome (hg19). It was chosen among approximately 1000 other randomly generated target sequences due to its high optimization score ( > 98%) and high expression in an initial screen containing iRFP (figure 1).

The target sequence is expressed by a guide RNA (gRNA) expression vector (BBa_K1875011) from the BostonU Gemini Library. The gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter (BBa_K1875014) to activate a downstream gene of interest (figure 2).

Figure 1: Initial screen of BostonU's first 18 operator reporters containing GFP

Figure 2: [http://2016.igem.org/Team:BostonU BostonU 2016] Project description

Sequence and Features